Metatranscriptomic profiling reveals diverse tick‐borne bacteria, protozoans and viruses in ticks and wildlife from Australia

Abstract Tick‐borne zoonoses are emerging globally due to changes in climate and land use. While the zoonotic threats associated with ticks are well studied elsewhere, in Australia, the diversity of potentially zoonotic agents carried by ticks and their significance to human and animal health is not sufficiently understood. To this end, we used untargeted metatranscriptomics to audit the prokaryotic, eukaryotic and viral biomes of questing ticks and wildlife blood samples from two urban and rural sites in New South Wales, Australia. Ixodes holocyclus and Haemaphysalis bancrofti were the main tick species collected, and blood samples from Rattus rattus, Rattus fuscipes, Perameles nasuta and Trichosurus vulpecula were also collected and screened for tick‐borne microorganisms using metatranscriptomics followed by conventional targeted PCR to identify important microbial taxa to the species level. Our analyses identified 32 unique tick‐borne taxa, including 10 novel putative species. Overall, a wide range of tick‐borne microorganisms were found in questing ticks including haemoprotozoa such as Babesia, Theileria, Hepatozoon and Trypanosoma spp., bacteria such as Borrelia, Rickettsia, Ehrlichia, Neoehrlichia and Anaplasma spp., and numerous viral taxa including Reoviridiae (including two coltiviruses) and a novel Flaviviridae‐like jingmenvirus. Of note, a novel hard tick‐borne relapsing fever Borrelia sp. was identified in questing H. bancrofti ticks which is closely related to, but distinct from, cervid‐associated Borrelia spp. found throughout Asia. Notably, all tick‐borne microorganisms were phylogenetically unique compared to their relatives found outside Australia, and no foreign tick‐borne human pathogens such as Borrelia burgdorferi s.l. or Babesia microti were found. This work adds to the growing literature demonstrating that Australian ticks harbour a unique and endemic microbial fauna, including potentially zoonotic agents which should be further studied to determine their relative risk to human and animal health.


INTRODUCTION
Ticks are important disease vectors and can transmit a diverse array of infectious agents to their vertebrate hosts, including bacteria, protozoa, helminths and viruses (de la Fuente et al., 2008). Incidences of human tick-borne disease are increasing in many parts of the world due to climate change and changes in land use (Diuk-Wasser et al., 2020;Gilbert, 2021). While the risks posed by ticks as vectors of zoonotic disease are well studied in parts of Europe and North America, in Australia, the significance of ticks as vectors of emerging zoonoses is not well understood.
Australia has a unique tick fauna, most of which are endemic only to Australia and New Guinea and are evolutionarily distinct to their relatives elsewhere (Barker & Walker, 2014;Roberts, 1970). For example, Australian Ixodes, Amblyomma and Bothriocroton species ticks form deeply rooted monophyletic lineages that diverged from their overseas relatives 112-51 mya (Beati & Klompen, 2019;Charrier et al., 2018).
Although Australia's tick fauna is unique and diverse, few species are anthropophilic, with Ixodes holocyclus, Ixodes tasmani and Haemaphysalis bancrofti being the most common in eastern Australia (Barker & Walker, 2014;Kwak, 2018).
The emergence of tick-borne zoonoses is of longstanding community concern in parts of Australia, particularly in relation to a poorly described chronic illness that is historically associated with tick bites (Chalada et al., 2016;Collignon et al., 2016). While the association to tick-bites is predominantly anecdotal (Brown, 2018;Storey-Lewis, 2019), this illness has been termed Debilitating Symptom Complexes Attributed to Ticks (DSCATT) to acknowledge the multifaceted nature of the illness and to create clarity from other terminology such as 'Lyme-like disease' which had previously been used. Borrelia burgdorferi s.l., the aetiological agent of Lyme disease, has not been reliably identified in Australian ticks, wildlife, domestic animals or people (except for nonautochthonous infections diagnosed in Australia) (Chalada et al., 2016;Collignon et al., 2016;Egan, Loh, et al., 2020;Egan, Taylor, Austen, et al., 2021;Gofton et al., 2015;Harvey et al., 2019;Irwin et al., 2017). Indeed, compared to other continents, Australia has relatively few tick-borne human pathogens, including several endemic Rickettsia spp. (R. australis, R. honei and R. honei subsp. marmionii) (Dehhaghi et al., 2019). Other facultative tick-borne pathogens such as Coxiella burnetti, Burkholderia pseudomallei, Francisella tularensis holarctica and F. hispaniensis also occur in Australia; however, the relative importance of tick-borne transmission in the epidemiology of these pathogens is thought be minimal (or is undocumented) compared to other transmission routes (Dehhaghi et al., 2019).
Despite the relative paucity of tick-borne human pathogens in Australia, there appears to be a rich diversity of microorganisms carried by Australian ticks, and there is a growing body of work demonstrat-ing that Australian ticks and wildlife harbour unique microbial fauna, including some potentially zoonotic agents. For example, recent studies have identified novel Borrelia (Beard et al., 2021;Loh et al., 2016;Panetta et al., 2017), Ehrlichia (Gofton et al., , 2018, Neoehrlichia (Egan, Loh, et al., 2020;Gofton et al., 2016), Babesia (Greay et al., 2018;Loh et al., 2018;Storey-Lewis et al., 2018), Iflaviridae (O'Brien et al., 2018) and Reoviridae (Harvey et al., 2019) in Australian ticks and wildlife. However, it should be noted that studies seeking to clarify the zoonotic potential of these microorganisms are currently lacking.
Metagenomics has emerged as a powerful tool for disease surveillance due to the technology's ability to identify taxonomically diverse and uncharacterised microorganisms in a wide range of sample types without a priori hypotheses. While metatranscriptomics has primarily been used for functional microbiome profiling or for the detection and characterisation of viruses, it has the power to detect and classify RNA molecules from all domains of life (Batson et al., 2021;Larsen et al., 2016;Marcelino et al., 2019;Ortiz-Baez et al., 2020;Westreich et al., 2019). To this end, we utilised metatranscriptomic sequencing to simultaneously investigate the prokaryotic, eukaryotic, and viral biomes of questing ticks and wildlife blood samples with the aim of identifying novel potentially zoonotic tick-borne microorganisms at two study sites in coastal New South Wales (NSW), Australia.

Tick and wildlife sampling
Ticks and wildlife samples were collected from October 2019 to December 2020 from two study areas, one located on the Northern Beaches area of northern Sydney, (NSW), Australia, and one located in the township of Kioloa, on the mid-south coast of NSW, Australia ( Figure 1). Sites were in remnant urban bushland or urban-adjacent rural bushland. At each sampling event, questing ticks were collected over a 3-day period by dragging a 1 m 2 white flannel cloth across leaf litter and over low-lying vegetation (Newman et al., 2019;Simmons et al., 2021) and removing tick with forceps into vials. Questing ticks were frozen and stored in the field on dry ice before being stored at −80 • C within 3 days of collection. Ticks were morphologically identified to species, life stage, and sex using standard keys (Barker & Walker, 2014;Roberts, 1970

PCR confirmation of novel tick-associated microorganisms
PCR and qPCR assays were used to confirm the detection of selected novel microorganisms and screen for known tick-borne pathogens in all pooled tick samples and individual wildlife blood samples. PCR primers, probes and assay conditions are provided in Table S1. PCR products were electrophoresed through 1-2% agarose gels and positive PCR products excised, purified and Sanger sequenced using both 5′ and 3′ PCR primers. Forward and reverse chromatograms were aligned with MAFFT (Katoh et al., 2002) in Geneious Prime v2020.2.5 (Kearse et al., 2012) before being aligned to reference sequences with MAFFT. Maximum likelihood phylogenetic analyses were performed in IQ-TREE (Minh et al., 2020) with model selection (Kalyaanamoorthy et al., 2017) and 5000 bootstrap replicates (Hoang et al., 2018). The prevalence of tick-borne microorganisms in variable-sized tick pools was calculated using maximum likelihood estimation according to Williams and Moffitt (2001).

Sample collection
A total of 3665 questing ticks were collected from field sites ( Figure 1 were the dominant species trapped (n = 11). Perameles nasuta and T.
vulpecula were trapped at both field sites; however, P. nasuta were more common in northern Sydney (n = 18) compared to Kioloa (n = 1), while equal numbers (n = 3) of T. vulpecula were trapped in northern Sydney and Kioloa.
Filtering of host and unassigned contigs removed an average of 99.33% and 68.48% of contigs from wildlife blood and tick samples, respectively, leaving 2163 unique taxonomic classifications of which 1996 were assigned to at least a family-level taxonomy.
Alpha diversity calculations showed no significant difference (p > .01) in microbial diversity levels between tick species, site, or between different life stages between or within species (Figures S1, S2). When grouped together, wildlife blood samples had significantly less (p < .01) microbial diversity compared to tick samples, but there was no significant (p > .01) difference in diversity levels between wildlife species, although some R. rattus and T. vulpecula samples had elevated microbial diversity compared to other samples ( Figure S2).
PCoA ordination of beta diversity demonstrated that tick species

Tick-borne Rickettsiales bacteria
In addition to endosymbiotic Rickettsia sp. found in H. bancrofti, Rickettsia contigs were also present in four I. holocyclus samples (MRA: 0.04%), including two nymph pools and one female pool from Kioloa, and one nymph pool from northern Sydney (  Figure S5). Herein, we refer to this species as Borrelia sp. HB, designated after its putative tick vector H. bancrofti.

Apicomplexan parasites in ticks and wildlife
The protozoan families Babesiidae, Theileriidae and Hepatozoidae contain only one genus each (Babesia, Theileria and Hepatozoon, respectively) which all contain tick-borne vertebrate-infecting parasites and were among the most highly abundant and prevalent taxa identified ( Figure 3, banethi ( Figure S7).

3.6
Trypanosoma spp. in ticks in wildlife  Figure S4). These contigs were exclusively found in nymph samples and only one species, T. gilletti, was identified in all I. holocyclus samples (98.9-99.9% 18S similarity) ( Figure S9).  18S similarity, respectively) and phylogenetically clustered with these species ( Figure S9). Unsuccessful attempts were made to amplify Trypanosoma gGAPDH sequences for further phylogenetic characterisation using the protocols of Hamilton et al. (2004) and McInnes et al. (2009), and no gGAPDH contigs were found in the metatranscriptomic data.

Tick-associated viruses
The complete or partial genomes of 12 viruses were identified in tick samples ( to Twyford virus and several Hubei picorna-like viruses from Dipteran hosts ( Figure 6).

DISCUSSION
In this study, we utilised metatranscriptomic sequencing to simultaneously interrogate the prokaryotic, eukaryotic and viral biomes of questing tick and wildlife blood samples to provide a comprehensive audit of potentially zoonotic tick-borne microorganisms in two sites in coastal New South Wales (NSW), Australia. We identified a wide range of taxonomically diverse vertebrate-infecting microorganism in questing ticks including several potentially zoonotic agents such as Borrelia sp., coltiviruses and jingmenviruses. In addition, our results add to the growing literature demonstrating that Australian ticks and their wildlife hosts harbour a unique and endemic microbial diversity that is largely distinct from the tick-borne pathogens found on other continents and concurs with previous reports that found no evidence of B. burgdorferi s.l. or other northern hemisphere tick-borne pathogens in Australian ticks or wildlife.
Borrelia spp. have previously been identified in Australian Bothriocroton spp. ticks and their wildlife hosts including echidnas (Tachyglossus aculeatus) and B. concolor (Loh et al., 2016); lace monitors (Varanus varius) and B. undatum (Panetta et al., 2017); and wombats (Vombatus ursinus) and B. auruginans (Beard et al., 2021). There is little evidence that these Borrelia spp. are transmitted by anthropophilic tick species (such as I. holocyclus), and it is probable that their host range is constrained by the strict host associations of their putative primary Both- More recently, a novel spirochete closely related to putative Borrelia sp. R57 was identified for the first time in Australia in R. rattus . This spirochete belongs to a unique rodentassociated lineage that is likely globally dispersed by anthropogenic rodents Fedorova et al., 2014;H. Gil et al., 2005). The absence of these spirochetes in R. rattus blood samples in this study could be explained by the apparent tissue-specific tropism of these spirochetes H. Gil et al., 2005).  (Furuno et al., 2017;Lee et al., 2014), H. hystricis in Malaysia (Khoo et al., 2017), H. longicornis in China  and Haemaphysalis spp. in Laos (Taylor et al., 2016). These Borrelia sp. have also been associated with cervid hosts including Sika deer (Cervus nippon) in Japan and Père David's Deer (Elaphurus davidianus) in China, as well as wild boar (Sus scrofa) and racoons (Procyon lotor) in Japan (Furuno et al., 2017;Kumagai et al., 2018;Yang et al., 2018).
The vertebrate hosts of Borrelia sp. HB were not identified in this study, and H. bancrofti has a wide host range with the majority of records from Macropodidae spp., cattle (Bos taurus and B. indicus) and humans (Barker & Walker, 2014;Laan et al., 2011). While there is limited evidence that H. bancrofti frequently parasitizes deer or wild boars in Australia (Laan et al., 2011;McKenzie et al., 1985), comprehensive surveys of deer and wild boar ectoparasites have not been conducted in Australia, and the level of parasitism by H. bancrofti could be underestimated. Therefore, a broad range of potential hosts including native and introduced species should be considered in future studies aiming to identify the vertebrate hosts of Borrelia sp. HB. Given the well-known pathogenic potential of other HTRF Borrelia spp. (Telford et al., 2015), this novel species warrants further ecological and epidemiologic investigation to better understand its potential pathogenic risks.
The detection here of Rickettsia australis, Candidatus Neoehrlichia australis, Candidatus Neoehrlichia arcana and Anaplasma bovis was not unexpected, as these endemic tick-borne bacteria have been reported previously (Egan, Loh, et al., 2020;Gofton et al., 2016;Hussain-Yusuf et al., 2020). R. australis is the only human pathogen identified in this study, with the prevalence in questing I. holocyclus estimated to be 1.4% (95%CI = 0.3-3.5%) in Kioloa and 2.1% (95%CI = 0.1-8.9%) in northern Sydney. This is lower than other studies from northern NSW and Queensland that found infections prevalence as high as 15.4% (Graves et al., 2016;Hussain-Yusuf et al., 2020); however, disparities in environment and reservoir host populations likely account for these differences.
NTV and OHV were identified here for the first time and phylogenetically cluster within the jingmenvirus (Flaviviridae-like) and coltivirus (Reoviridae) genera, respectively ( Figure 6). Another novel coltivirus, Shelly Headland virus, was also recently described from a limited number of I. holocyclus from NSW and was also detected here in I. holocyclus samples at a much higher prevalence than OHV ( Although Longdon et al. (2015) suggests that most dimarhabdoviruses, including the clade that contains KTV, are arboviruses, no tickassociated dimarhabdoviruses have ever been found in vertebrate hosts, with the study also concluding that rhabdovirus host switching events between more distantly related hosts is rare. SV contigs found in H. bancrofti samples were highly similar to several Hubei picorna-like viruses ( Figure 6) that infect Entomophthora muscae, a behaviour-manipulating fungal pathogen of dipterans (Coyle et al., 2018). Ticks are susceptible to entomopathogenic fungal pathogens such as Metarhizium spp. (Goettel et al., 2005), and it is probable that SPV is associated with such a fungus.  (Egan, Taylor, Austen, et al., 2020;Egan, Taylor, Austen, et al., 2021;Greay et al., 2018 Although we attempted to conduct our field sampling in a comprehensive manner, unavoidable disruptions from bushfires and SARS-CoV-2 travel restrictions severely hampered our efforts to systematically survey the tick and wildlife fauna at field sites. For this reason, the samples collected during this study may not accurately represent the tick or wildlife populations at each field site (indeed, Macropodidae species which are common in Sydney and Kioloa, NSW were not sampled at all).
Nevertheless, even with modest sampling at few localities, we identified a remarkably abundant and diverse array of vertebrate-infecting tick-borne microorganisms, several of which may have zoonotic potential. This outcome is in agreement with recent studies investigating Australian ticks, and collectively, these works convincingly highlight both the richness of Australia's tick-borne microflora and how underexplored this area is, particularly in relation to zoonotic agents (Chandra et al., 2021;Egan, Loh, et al., 2020;Egan, Taylor, Austen, et al., 2021;Gofton et al., 2015;Greay et al., 2018;Harvey et al., 2019). We expect therefore that more extensive sampling of diverse ticks and wildlife across Australia would uncover additional diversity and further novel tick-borne microorganisms with zoonotic potential.
The distinction of this study compared to others applying metatranscriptomic techniques to the exploration of tick-borne microorganisms is that our taxonomically impartial analysis allowed the identification of bacterial, protozoan and viral microorganisms without prejudice.
Typically, metatranscriptomic studies are limited to the exploration of viral taxa only, and although highly appropriate for such studies, this technology also offers opportunities for the exploration of other biomes, as demonstrated in this work and other recent studies (Batson et al., 2021;Marcelino et al., 2019;Ortiz-Baez et al., 2020). By removing taxonomic restrictions on the analysis of metatranscriptomic data, researchers can more accuratly investigate and characterise entire microbial communities rather than individual biomes in isolation, leading to the a deeper understanding of the members present, their interactions, and how they may affect phenotypic traits.
Unbiased disease surveillance within a One Health framework can provide a holistic perspective of pathogen diversity at the individual, host or landscape scale, and is a fundamental tool that enables us to identify and respond to novel emerging pathogens. This work identified a range of tick-borne bacteria, protozoa and viruses from questing anthropogenic ticks and wildlife blood samples including several potentially zoonotic taxa related to known agents of human and animal disease. These potentially zoonotic taxa, including Borrelia sp. HB, NTV and OHV, warrants further study to clarify their pathogenic potential.
Although this work cannot implicate these novel tick-borne microorganisms as causes of disease, it does broaden our understanding of the richness of potentially zoonotic microbes associated with I. holocyclus and H. bancrofti and offers a springboard for further research to quantify, and hopefully mitigate, their zoonotic risk before potential disease emergence.

CONFLICT OF INTEREST
The author declares that there is no conflict of interest.

ETHICS APPROVAL
The authors confirm that the ethical policies of the journal have been adhered to and the appropriate ethical review committee approval has been received. The Australian Code for the Care and Use of Animals for Scientific Purposes were followed.

AUTHOR CONTRIBUTIONS
Alexander W. Gofton